The cell surface glycolipids (lipooligosaccharides, LOS) of Campylobacter jejuni show considerable structural diversity, with many ganglioside mimics being found in pathogenic strains and this has been correlated to the genetic diversity of the locus (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). The LOS biosynthesis gene clusters of a large number of C. jejuni strains have been examined and have been divided in eight classes (“A” to “H”) based on their gene content and genetic organization (Parker, C. T. et al., J Clin Microbiol. 43:2771-2781 (2005)). The first three classes (“A”, “B” and “C”) contain the neuBCA and cst-II genes needed for the biosynthesis of sialic acid and its incorporation into the growing LOS to constitute ganglioside mimics (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002); Parker, C. T. et al., J Clin Microbiol. 43:2771-2781 (2005)). As we are interested in the synthesis of sialylated glycans, we have focused our research efforts on the enzymes encoded by genes from clusters belonging to classes “A”, “B” and “C” (described in Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)).
There exists considerable amino acid sequence variation between C. jejuni strains for the same LOS biosynthesis glycosyltransferase (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). The investigation of the LOS biosynthesis gene clusters from eleven C. jejuni strains expressing eight different serotypes led to the identification of five distinct mechanisms by which this bacterium can vary the structure of its LOS (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). One of these mechanisms consists in the occurrence of single or multiple mutations leading to “allelic” glycosyltransferases with different acceptor specificities (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). This was clearly illustrated by the investigation of variants of CgtA (β1,4-N-acetylgalactosaminyltransferase) and of Cst-II (α-2,3-/2,8-sialyltransferase). Six CgtA variants (sharing 34% overall amino acid sequence identity) from classes “A”, “B” and “C” displayed different specific activities towards non-sialylated (lactose), mono (GM31)- or disialylated (GD31) FCHASE-labeled acceptors (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). Similarly, six variants of Cst-II (sharing 92% overall amino acid sequence identity) from classes “A” and “B” displayed different specific activities towards non-sialylated (lactose) or sialylated (GM31) FCHASE-labeled acceptors (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). A Cst-II variant belonging to class “C” had previously been shown to be active with lactose-FCHASE and inactive with GM3-FCHASE (Gilbert, M et al., J Biol. Chem. 275:3896-3906 (2000)). These differences in acceptor preference and in specific activity levels are a consequence of amino acid sequence divergence between enzyme variants.
As is the case for CgtA, CgtB (β1,3-galactosaminyltransferase) enzymes exhibit sequence divergence: only 47% amino acid sequence identity has been reported between the sequences from eleven strains belonging to classes “A”, “B” and “C” (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). The activity and acceptor preference of CgtB variants from classes “A”, “B” and “C” had not been determined, however, as has been done for CgtA and Cst-II (Gilbert, M et al., J Biol. Chem. 275:3896-3906 (2000); Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)).
The CgtB sequences from strains ATCC 43432 (SEQ ID NOs: 3 and 4), OH4384 (SEQ ID NOs: 1 and 2) and ATCC 43460 (SEQ IDs: 5 and 6) (serotypes HS:4, HS:19 and HS:41, respectively; all members of class “A”) share 99% sequence identity (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). CgtB from strain OH4384 (SEQ ID NOs: 7 and 8) (CgtBOH4384, also of serotype HS:19; FIG. 1A) was chosen for further characterization since it had already been partially characterized (Gilbert, M et al., J Biol. Chem. 275:3896-3906 (2000)). CgtB from strain NCTC 11168 (SEQ ID NOs: 7 and 8) (CgtB11168; serotype HS:2; FIG. 1B), a member of the class “C” group was also chosen as it has been used for the in vitro synthesis of the glycone moiety of ganglioside GM1a and for the synthesis of ganglioside mimics (Linton, D. et al., Mol. Microbiol. 37:501-14 (2000); Blixt, O. et al., Carbohydrate Res. 340:1963-1972 (2005)). A second member of class “A”, that from strain ATCC 43438 (SEQ ID NOs: 9 and 10) (CgtBHS:10; serotype HS:10), was included for the comparison because its sequence differs from those of the other members of class “A”. Compared to the other members of its class, the carboxy-terminal of CgtBHS:10 contains a large number of amino acid substitutions (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)). These changes may reflect acceptor preferences (FIG. 1C; Gilbert, M. et al., J Biol Chem. 277:327-337 (2002)). No CgtB variant from class “B” was chosen, as their amino acid sequences are not distinct from those of class “A” (Gilbert, M. et al., J Biol. Chem. 277:327-337 (2002)).
Bacterial glycosyltransferases have been successfully incorporated in various chemo-enzymatic schemes. Improved enzymatic activities could increase the use of bacterial glycosyltransferases in production of oligosaccharides including glycoproteins, glycopeptides, glycolipids, and gangliosides.